The development of an effective vaccine for a number of viral and bacterial pathogens is a substantial unmet human health need. In particular, there is a need for human immunodeficiency virus type-1 (HIV-1) vaccine (Johnston et al., “An HIV Vaccine—Challenges and Prospects,” N Engl J Med. 359(9):888-90 (2008), Barouch et al., “Challenges in the Development of an HIV-1 Vaccine,” Nature 455(7213):613-9 (2008)). A major roadblock in this process is the present inability to elicit broadly neutralizing antibodies (BNA) that recognize the HIV-1 envelope, following immunization with candidate HIV-1 vaccines. This is believed to reflect, in part, the sequestration of key neutralizing epitopes on HIV-1 Env (Pantophlet, “GP120: Target for Neutralizing HIV-1 Antibodies,” Annu Rev Immunol. 24:739-69 (2006); Walker, “Toward an AIDS Vaccine,” Science 320(5877):760-4 (2008)).
The surface HIV-1 envelope glycoprotein subunit, gp120, is a major target for virus-neutralizing antibodies (Pantophlet, “GP120: Target for Neutralizing HIV-1 Antibodies,” Annu Rev Immunol. 24:739-69 (2006)), but immunization with recombinant gp120 failed to elicit broadly neutralizing antibodies and failed to demonstrate protective efficacy in a Phase III clinical trial (Gilbert et al., “HIV-1 Virologic and Immunologic Progression and Initiation of Antiretroviral Therapy Among HIV-1-infected Subjects in a Trial of the Efficacy of Recombinant Glycoprotein 120 Vaccine,” J Infect Dis. 192(6):974-83 (2005); Gilbert et al., “Correlation Between Immunologic Responses to a Recombinant Glycoprotein 120 Vaccine and Incidence of HIV-1 Infection in a Phase 3 HIV-1 Preventive Vaccine Trial,” J Infect Dis. 191(5):666-77 (2005)). Other approaches have attempted to use more native (oligomeric) forms of the envelope spike and/or re-engineered forms of the HIV-1 envelope, designed to display otherwise sequestered domains that may serve as targets for BNA. However, these approaches have yet to elicit the desired antibody response (Herrera et al., “The Impact of Envelope Glycoprotein Cleavage on the Antigenicity, Infectivity, and Neutralization Sensitivity of Env-pseudotyped Human Immunodeficiency Virus Type 1 Particles,” Virology 338(1):154-72 (2005) and Poignard et al., “Heterogeneity of Envelope Molecules Expressed on Primary Human Immunodeficiency Virus Type 1 Particles as Probed by the Binding of Neutralizing and Normeutralizing Antibodies,” J Virol. 77(1):353-65 (2003)).
HIV-1 Env presents a recalcitrant antigenic target in part because of the extensive glycosylation that hides antibody epitopes, and also because conserved domains which can serve as potential targets for broadly neutralizing antibodies are either physically sequestered or represent transient conformational intermediates (Wei et al., “Antibody Neutralization and Escape by HIV-1,” Nature 422(6929):307-12 (2003); Wyatt et al., “The Antigenic Structure of the HIV gp120 Envelope Glycoprotein,” Nature 393(6686):705-11 (1998); Rizzuto et al., “A Conserved HIV gp120 Glycoprotein Structure Involved in Chemokine Receptor Binding,” Science 280(5371):1949-53 (1998); and Kwong et al., “Structure of an HIV gp120 Envelope Glycoprotein in Complex with the CD4 Receptor and a Neutralizing Human Antibody,” Nature 393(6686):648-59 (1998), which are hereby incorporated by reference in their entirety). These considerations have led to attempts to rationally design improved Env immunogens that are better able to induce broadly neutralizing antibodies, and to create synthetic mimics of neutralizing antibody epitopes (Pantophlet, “GP120: Target for Neutralizing HIV-1 Antibodies,” Annu Rev Immunol. 24:739-69 (2006); Saphire et al., “Crystal Structure of a Neutralizing Human IGG Against HIV-1: a Template for Vaccine Design,” Science 293(5532):1155-9 (2001); Calarese et al., “Dissection of the Carbohydrate Specificity of the Broadly Neutralizing Anti-HIV-1 Antibody 2G12,” Proc Natl Acad Sci USA 102(38):13372-7 (2005); Zwick et al., “Identification and Characterization of a Peptide that Specifically Binds the Human, Broadly Neutralizing Anti-human Immunodeficiency Virus Type 1 Antibody b12,” J Virol. 75(14):6692-9 (2001); Ni et al., “Toward a Carbohydrate-based HIV-1 Vaccine: Synthesis and Immunological Studies of Oligomannose-containing Glycoconjugates,” Bioconjug Chem. 17(2):493-500 (2006); Pashov et al., “Antigenic Properties of Peptide Mimotopes of HIV-1-associated Carbohydrate Antigens,” J Biol Chem. 280(32):28959-65 (2005); Pashov et al., “Multiple Antigenic Mimotopes of HIV Carbohydrate Antigens—relating Structure and Antigenicity,” J Biol Chem. (281): 29675-29683 (2006)). However, such an immunogen has not been developed.
There is also a need for new influenza virus vaccines to fight emerging avian influenza A viruses. The 2009 H1N1 influenza A virus (IAV) epidemic underscores the ability of antigenically novel IAV strains to rapidly infect human populations, creating the potential for a new viral pandemic. This same epidemic confirmed that conventional methods of generating the IAV vaccine are unsuitable for immediate response to the pathogen.
Highly pathogenic avian influenza A viruses that possess a new H5 subtype of hemagglutinin have been linked to numerous instances of human transmission, resulting in severe disease or death (Beigel et al., “Avian Influenza A (H5N1) Infection in Humans,” N. Engl. J. Med. 353:1374-85 (2005); Tran et al., “Avian Influenza A (H5N1) in 10 Patients in Vietnam,” N. Engl. J. Med. 350:1179-88 (2004)). There is considerable concern with regard to the pandemic potential of these viruses, due to the lack of H5-specific immunity in human populations and the widespread presence of virus in bird populations throughout Asia, Africa and Europe (Li et al., “Genesis of a Highly Pathogenic and Potentially Pandemic H5N1 Influenza Virus in Eastern Asia,” Nature 430:209-13 (2004); Olsen et al., “Global Patterns of Influenza a Virus in Wild Birds,” Science 312:384-8 (2006)). Moreover, resistance of these strains to existing antiviral drugs such as oseltamivir has been described (de Jong et al., “Oseltamivir Resistance During Treatment of Influenza A (H5N1) Infection,” N. Engl. J. Med. 353:2667-72 (2005)). While human-to-human transmission of these highly pathogenic avian influenza virus strains appears to be rare (Ungchusak et al., “Probable Person-to-Person Transmission of Avian Influenza A (H5N1),”N Engl J Med 352:333-40 (2005)), there is a pressing need to develop new vaccines against influenza A (H5N1) virus.
Human clinical trials have shown that baculovirus-expressed recombinant hemagglutinins (rHA) can elicit serum antibody responses in both healthy and elderly adults (Lakey et al., “Recombinant Baculovirus Influenza A Hemagglutinin Vaccines Are Well Tolerated and Immunogenic in Healthy Adults,” J. Infect. Dis. 174:838-41 (1996); Treanor et al., “Evaluation of a Recombinant Hemagglutinin Expressed in Insect Cells as an Influenza Vaccine in Young and Elderly Adults,” J. Infect. Dis. 173:1467-70 (1996); Treanor et al., “Dose-Related Safety and Immunogenicity of a Trivalent Baculovirus-Expressed Influenza-Virus Hemagglutinin Vaccine in Elderly Adults,” J. Infect. Dis. 193:1223-8 (2006); Treanor et al., “Safety and Immunogenicity of a Recombinant Hemagglutinin Vaccine for H5 Influenza in Humans,” Vaccine 19:1732-7 (2001)). However, because the HA is administered as a soluble protein without adjuvant, relatively high doses have been required to achieve protective immunity; this has been a particular problem for the H5 rHA (Treanor et al., “Safety and Immunogenicity of a Recombinant Hemagglutinin Vaccine for H5 Influenza in Humans,” Vaccine 19:1732-7 (2001)), as well as for egg-derived H5 vaccines in humans (Treanor et al., “Safety and Immunogenicity of an Inactivated Subvirion Influenza A (H5N1) Vaccine,” N. Engl. J. Med. 354:1343-51 (2006)). Moreover, current data suggest that alum, the most widely available adjuvant for vaccines in humans, will not have a significant dose-sparing effect for H5 vaccines in man (Bresson et al., “Safety and Immunogenicity of an Inactivated Split-Virion Influenza A/Vietnam/1194/2004 (H5N1) Vaccine: Phase I Randomized Trial,” Lancet: published online May 11, 2006 DOI:10.1016/S0140-6736(06)68656-X (2006); Powers et al., “Influenza A Virus Vaccines Containing Purified Recombinant H3 Hemagglutinin Are Well Tolerated and Induce Protective Immune Responses in Healthy Adults,” J. Infect. Dis. 171:1595-9 (1995)).
An inactivated subvirion vaccine developed by Sanofi has recently been evaluated in a human clinical trial. This product induces protective antibody levels in only 45% of recipients, even when used at a high dose, but was nonetheless recently recommended for licensure by VRBPAC as a “stop-gap” measure. This underscores the need to develop alternative approaches to increase the immunogenicity of H5 vaccines, and decrease the dose needed to achieve protective immunity—such that large-scale production will become feasible.
Monoclonal antibodies with broadly-neutralizing (BN) activity against group 1 influenza viruses have recently been identified, and shown to react against a conserved conformational epitope located in the stem of the hemagglutinin (HA) ectodomain, thereby preventing membrane fusion. Broad spectrum antibodies of this type are not, however, generated during influenza virus infection or immunization in humans. This is thought to be a consequence of an immunodominant response to the exposed globular head domain of HA.
It would be desirable, therefore, to generate an antigenic mimic of discontinuous pathogen epitopes that are recognized by broadly neutralizing antibodies, and which can be directly administered to an individual to induce an immune response against the pathogen of interest, or to bind to unmutated genomic antibody genes capable of either directly neutralizing the infectivity of a pathogen of interest or giving rise to broadly neutralizing antibodies.
The present invention overcomes these and other deficiencies in the art.